Reversing protonation of weakly basic drugs greatly enhances intracellular diffusion and decreases lysosomal sequestration

Reviewer #1 (Public Review):

Summary:

The authors set out to measure the diffusion of small drug molecules inside live cells. To do this, they selected a range of flourescent drugs, as well as some commonly used dyes, and used FRAP to quantify their diffusion. The authors find that drugs diffuse and localize within the cell in a way that is weakly correalted with their charge, with positively charged molecules displaying dramatically slower diffusion and a high degree of subcellular localization.
The study is important because it points at an important issue related to the way drugs behave inside cells beyond the simple "IC50" metric (a decidedly mesoscopic/systemic value). The authors conclude, and I agree, that their results point to nuanced effects that are governed by drug chemistry that could be optimized to make them more effective.

Strengths:

The work examines an understudied aspect of drug delivery.
The work uses well-established methodologies to measure diffusion in cells
The work provides an extensive dataset, covering a range of chemistries that are common in small molecule drug design
The authors consider several explanations as to the origin of changes in cellular diffusion

Weaknesses:

The results are described qualitatively, despite quantitative data that can be used to infer the strength of the proposed correlations.
The statistical treatment of the data is not rigorous and not visualized according to best practices, making it difficult for readers to assess the significance of the findings.
Some important aspects of drug behavior are not discussed quantitatively, such as the cell-to-cell or subcellular variability in concentration.
It is unclear if the observed behavior of each drug in the cell actually relates to its efficacy - though this is clearly beyond the scope of this specific work.

Reviewer #2 (Public Review):

Summary: Blocking a weak base compound's protonation increased intracellular diffusion and fractional recovery in the cytoplasm, which may improve the intracellular availability and distribution of weakly basic, small molecule drugs and be impactful in future drug development.

Strengths:

1. The intracellular distribution of drugs and the chemical properties that drive their distribution are much needed in the literature. Thus, the idea behind this paper is of relevance.

2. The study used common compounds that were relevant to others.

3. Altering a compound's pKa value and measuring cytosolic diffusion rates certainly is inciteful on how weak base drugs and their relatively high pKa values affect distribution and pharmacokinetics. This particular experiment demonstrated relevance to drug targeting and drug development.

4. The manuscript was fairly well written.

Weaknesses:

1. Small sample sizes. 2 acids and 1 neutral compound vs 6 weak bases (Figure 1).

2. A comparison between the percentage of neutral and weak base drug accumulation in lysosomes would have helped indicate weak base ion trapping. Such a comparison would have strengthened this study.

3. When cytosolic diffusion rates of compounds were measured, were the lysosomes extracted from the image using Imaris to determine a realistic cytosolic value? In real-time, lysosomes move through the cytosol at different rates. Because weak base drugs get trapped, it is likely the movement of a weak base in the lysosome being measured rather than the movement of a weak base itself throughout the cytosol. This was unclear in the methods. Please explain.

4. Because weak base drugs can be protonated in the cytoplasm, the authors need to elaborate on why they thought that inhibiting lysosome accumulation of weak bases would increase cytosolic diffusion rates. Ion trapping is different than "micrometers per second" in the cytosol. Moreover, treating cells with sodium azide de-acidifies lysosomes and acidifies the cytosol; thus, more protons in the cytosol means more protonation of weak base drugs. The diffusion rates were slowed down in the presence of lysosome inhibition (Figure 7), which is more fitting of the story about blocking protonation increases diffusion rates, but in this case, increasing cytosolic protonation via lysosome de-acidification agents decreases diffusion rates. Please elaborate.

A discussion of the likely impact:
The manuscript certainly adds another dimension to the field of intracellular drug distribution, but the manuscript needs to be strengthened in its current form. Additional experiments need to be included, and there are clarifications in the manuscript that need to be addressed. Once these issues are resolved, then the manuscript, if the conclusions are further strengthened, is much needed and would be inciteful to drug development.